Declines in the mitochondrial oxidative phosphorylation (OXPHOS) system are associated with bona fide mitochondrial diseases as well as with numerous neurodegenerative disorders (e.g. Parkinson's, Alzheimer's and Huntington's disease). Currently, there is no cure for any of the diseases involving OXPHOS dysfunction. The OXPHOS system is comprised of the respiratory complexes of the electron transport chain (complexes CI to CIV) and CV. Defects in OXPHOS function can cause elevation of the levels of reactive oxygen species (ROS), a common occurrence in many neurological disorders. It is believed that increased ROS contributes immensely to the neuronal pathology. The OXPHOS complexes can interact with each other to form supramolecular structures named supercomplexes. These structures are thought to optimize electron transfer and minimize the escape of reactive intermediates. Defects in one respiratory complex can affect the stability of another and in turn affects the capacity of forming supercomplexes. Currently, it is not known how supercomplexes are regulated and what role they have in health and disease. We have recently found that CI, CIV and supercomplexes assemblies are unstable in cells with defective CIII. Moreover, we discovered that this effect could be reversed by low concentrations of oxygen (1% O2). We hypothesize that the stabilizing effect of hypoxia is mediated by a decrease in the levels of ROS. If this is correct it implies that oxygen levels or ROS can be physiological regulators of respiratory supercomplexes. We plan to test this hypothesis by accomplishing three aims: Aim 1 will test whether different types of OXPHOS defects show the same behavior we found in the complex III deficient cells. We have unique cell lines and mice tissue with OXPHOS defects to investigate the stabilizing effect of hypoxia, how long this effect last and its functional consequences. Aim 2 will confirm whether the hypoxia effect is mediated by ROS. Finally, Aim 3 will explore the mechanisms of hypoxia/ROS mediated stabilization of respiratory complexes and supercomplexes. Discerning the molecular basis of the hypoxia-induced stability of mitochondrial complexes and supercomplexes and understanding how respiratory complexes are regulated and how they respond to changes in oxygen levels and bioenergetics crises will open a new window for therapeutic intervention for many neurodegenerative diseases. Learning how to regulate respiratory complex and supercomplex assemblies will help us devise approaches to minimize free radical formation and further oxidative stress when cells are faced with a bioenergetic or oxygen challenges. PUBLIC HEALTH RELEVANCE: In recent years strong evidence indicates an important role for mitochondrial dysfunction in the pathology of many neurodegenerative and mitochondrial diseases. Defects in the respiratory chain lead to increased levels of free radical contributing to the oxidative stress produced in disease conditions. This study investigates the role and mechanisms of a newly discovered effect of hypoxia in the stabilization of mitochondrial complexes and supercomplexes (functional respiratory units) during impaired mitochondrial respiration. We hypothesize that regulating supercomplexes structures can minimize the production of free radicals avoiding further oxidative damage.